Detection and identification of enteric pathogens

ABSTRACT

The present invention is directed to methods and reagents for the specific detection and presumptive identification of various bacteria associated with waterborne infectious disease. In particular, this invention relates to methods and reagents for the specific detection and identification of Salmonella and Shigella in environmental samples such as water and sewage.

This is a divisional of copending application Ser. No. 08/232,778 filedon Apr. 25, 1994.

FIELD OF THE INVENTION

The present invention relates to methods and reagents for the detectionof enteric pathogens in environmental samples such as water, wastewater,sewage and sludge, as well as food, feed and clinical samples.

BACKGROUND OF THE INVENTION

An ever-enlarging world population has increased demands on waterresources worldwide. Indeed, this population increase is directlyproportional to the potential for surface and ground water contaminationby pathogenic organisms associated with increased waste burdens. Toensure good public health, there is a need for readily available methodsto detect and enumerate pathogens in water. Unfortunately, despite yearsof testing and research, no single procedure is available for thereliable detection of the major waterborne pathogens. Indeed, there areno standardized methods for detecting all of these pathogens. Themethods that are available are usually time-consuming and expensive.

Routine or periodic monitoring of water for the presence of pathogens isessential in situations such as wastewater reclamation, during and afterwaterborne outbreaks, and for water sources with a frequent history ofcontamination. This is largely due to the observation that most entericpathogens appear intermittently and in low concentrations in aquaticenvironments. Thus, potentially pathogenic organisms may be present in awater supply and go undetected, largely due to their low numbers and thelimitations of current testing methods, including relatively lowsensitivity levels.

Despite advances in public health technology, water and food remainimportant reservoirs of diarrheal and other diseases of humans and otheranimals. Infectious represents a significant public health concern.According to one estimate, infectious diarrhea results in thehospitalization of 200,000 children in the United States each year, atan annual cost of one billion dollars (M. Ho et al., "Rotavirus as acause of diarrheal morbidity and mortality in the United States," J.Infect. Dis., 158:1112-1116, 1988).

Worldwide, waterborne disease is of even greater significance, with over250 million reported cases of waterborne disease and more than 10million deaths annually (J. D. Snyder and M. H. Merson, "The magnitudeof the global problem of acute diarrheal disease: a review of activesurveillance data," Bull. World Health Organ., 60:605-613 [1982]). Whenother sources of diarrheal disease are taken into consideration thefigures are even more staggering, with these diseases claiming the livesof over 5 million children per year in developing countries (T. L. Hale,"Genetic basis of virulence in Shigella species," Microbiol. Rev.,55:206-224 [1991]).

Most of the cases of waterborne diarrheal disease result from thecontamination of drinking water supplies with human fecal material.Contamination of ground water in local areas may occur through suchmechanisms as seepage of sewage into aquifers and by improperlydeveloped or poorly protected wells. When factors such as recreationalexposure to contaminated salt and fresh water are also taken intoconsideration, diarrheal disease takes on even greater importance.

Various infectious agents are associated with human waterborne diseases,including Campylobacter, E. coli, Leptospira, Pasteurella, Salmonella,Shigella, Vibrio, Yersinia, Proteus, Giardia, Entoamoeba,Cryptosporidium, hepatitis A virus, Norwalk, parvovirus, polio virus,and rotavirus. Worldwide, the most common bacterial diarrheal diseasesare associated with waterborne transmission of Shigella, Salmonella,pathogenic E. coli, Campylobacter jejuni, and Vibrio cholerae (Singh andMcFeters, "Detection methods for waterborne pathogens," pp. 125-156, inR. Mitchell (ed.), Environmental Microbiology, [Wiley-Liss, New York,1992]). Table 1 lists important characteristics of diseases associatedwith a few of the most significant organisms.

                                      TABLE 1                                     __________________________________________________________________________    Waterborne Diarrheal Bacterial Diseases Most Commonly Reported                                    Incubation                                                Organism    Disease Period Common Symptoms                                    __________________________________________________________________________    Shigella sp.                                                                              Shigellosis                                                                           1-7 days                                                                             Diarrhea, fever, cramps, tenesmus, dysentery*      Salmonella typhimurium                                                                    Salmonellosis                                                                         6-72 hours                                                                           Abdominal pain, diarrhea, nausea,                                             vomiting, fever                                    S. typhi    Typhoid fever                                                                         1-3 days                                                                             Abdominal pain, fever, chills, diarrhea or                                    constipation, intestinal hemorrhage                Pathogenic E. coli                                                                        Diarrhea                                                                              12-72 hours                                                                          Diarrhea, fever, vomiting                          Campylobacter jejuni                                                                      Gastroenteritis                                                                       1-7 days                                                                             Abdominal pain suggesting acute                                               appendicitis, fever, headache, malaise,                                       diarrhea, vomiting                                 Proteus sp. Scombroid fish                                                                        Few minutes                                                                          Headache, dizziness, vomiting, nausea,                         poisoning                                                                             to 1 hour                                                                            peppery taste, burning throat, facial                                         swelling                                                                      and flushing, stomach pain, itching                Yersinia enterocolitica                                                                   Yersiniosis                                                                           24-36 hours                                                                          Severe abdominal pain, fever, headache             Vibrio parahaemo-lyticus                                                                          12 hours                                                                             Vomiting, diarrhea, abdominal pain, fever          Vibrio cholerae                                                                           Gastroenteritis                                                                       1-3 days                                                                             Vomiting, diarrhea, dehydration                    __________________________________________________________________________

Swimming-associated outbreaks caused by Shigella, Giardia, Norwalk-likeviruses, and other enteroviruses have been well documented [See e.g.,Makintubee et al., "Shigellosis outbreak associated with swimming," Am.J. Public Health 77:166-168 [1987]; F. J. Sorvillo et al., Shigellosisassociated with recreational water contact in Los Angeles County," Am.J. Trop. Med. Hyg., 38:613-617[1988]).

The following table lists the majority of waterborne infectious bacteriawhich are associated with human diarrheal and non-diarrheal disease.

                  TABLE 2                                                         ______________________________________                                        Infectious Bacteria Transmitted by Water                                                            Commonly                                                                      Associated                                              Organism              Diseases in Humans                                      ______________________________________                                        Acinetobacter calcoaceticus                                                                         Nosocomial infections                                   Aeromonas hydrophila  Enteritis,                                              A. sobria             wound infections                                        A. caviae                                                                     Campylobacter jejuni  Enteritis                                               C. coli                                                                       Chromobacterium violaceum                                                                           Enteritis                                               Citrobacter spp.      Nosocomial infections                                   Clostridium perfringens, type C                                                                     Enteritis                                               Enterobacter spp.     Nosocomial infections                                   E. coli, various serotypes                                                                          Enteritis                                               Flavobacterium meninogsepticum                                                                      Nosocomial infections,                                                        meningitis                                              Francisella tularensis                                                                              Tularemia                                               Fusobacterium necrophorum                                                                           Liver abscesses                                         Klebsiella spp.       Nosocomial infections,                                                        pneumonia                                               Leptospira icterohaemorrahagia                                                                      Leptospirosis                                           and other Leptospira spp.                                                     Legionella pneumophila                                                                              Legionellosis                                           and other Legionella spp.                                                     Morganella morganii   Urethritis, nosocomial                                                        infections                                              Mycobacterium tuberculosis                                                                          Tuberculosis                                            M. marinum and other Mycobacterium spp.                                                             Granuloma, dermatitis                                   Plesiomonas shigelloides                                                                            Enteritis                                               Pseudomonas pseudomallei                                                                            Melioidosis                                             Pseudomanas spp.      Dermatitis, ear                                                               infections                                              Salmonella enteritidis                                                                              Enteritis                                               S. montevideo B       (salmonellosis)                                         S. typhimurium                                                                and other Salmonella serotypes                                                S. paratyphi A and B  Paratyphoid fever                                       S. typhi              Typhoid fever                                           Serratia marcesens    Nosocomial infections                                   Shigella spp.         Dysentery                                               Staphylococcus aureus Wounds, food                                                                  poisoning                                               Vibrio cholerae       Cholera                                                 V. alginolyticus      Enteritis                                               V. fluvialis          Wound infections                                        V. mimicus                                                                    V. parahaemolyticus                                                           V. vulnificus                                                                 Other Vibrio spp.                                                             Yersinia enterocolitica                                                                             Enteritis                                               ______________________________________                                         *After T. C. Hazen and G. A. Toranzos, "Tropical Source Water," p. 33, in     G. A. McFeters, Drinking Water Microbiology [SpringerVerlag, New York,        1990]).                                                                  

While the presence of pathogens in drinking and recreational waterspresents a significant public health concern, recovery of pathogens fromenvironmental samples is generally difficult. In addition to the usuallylow numbers of organisms present in the water, nutrient limitations andenvironmental stressors produce unpredictable physiological andmorphological changes in these pathogens. This makes their isolation andidentification problematic. Organisms injured due to these environmentalstressors often exhibit atypical reactions and require specializedhandling for their resuscitation (see e.g., Singh and McFeters, p.132-133).

Often, organisms are present but are unculturable (Id., at 131-159; seealso, J. J. Byrd et al., "Viable but nonculturable bacteria in drinkingwater," Appl. Environ. Microbiol., 57:875-878 [1991]; C. Desmonts etal., "Fluorescent-antibody method useful for detecting viable butnonculturable Salmonella spp. in chlorinated wastewater," Appl. Environ.Microbiol., 56:1448-1442 [1990]; and J. J. Byrd and R. R. Colwell,"Maintenance of plasmids pBR322 and pUC8 in nonculturable Escherichiacoli in the marine environment," Appl. Environ. Microbiol., 56:2104-2107[1990]). Unless other methods are used for their detection (e.g.,immunoassays) these viable, but non-culturable organisms may present anundetected threat to public health.

In addition, the methods commonly used to detect these pathogens wereinitially designed for clinical, rather than environmental samples. Thisis of significance in view of the different ecological niche occupied byclinical as compared with environmental isolates. Clinical isolates areusually provided needed nutrients by their host animal and are generallyprotected from harsh environmental conditions such as cold, heat,damaging chemicals and radiation. In contrast, environmental isolatesmust deal with these environmental conditions and effectively competewith organisms naturally present and adapted to life in the environment.Pathogenic organisms are rarely readily adaptable to prolonged survivalin the environment. Thus, "indicator" organisms are used as a prognosticindication of whether pathogens may be present in a particular sample.

Use of Indicator Organisms to Detect Fecal Contamination of Water

Problems associated with recovery of pathogens from water led to thedevelopment of methods to detect and enumerate "indicators" of fecalcontamination. These organisms serve to indicate whether a given watersupply is contaminated with fecal material, without actually testing forthe presence of pathogens. This contamination is viewed as predictive ofthe potential presence of enteric pathogens (i.e., without the presenceof fecal material, the chances of these pathogens being present isusually remote). However, a number of issues remain to be resolved, notthe least of which is the significance of the presence of indicatororganisms in water supplies.

Criteria for the establishment of the "ideal" indicator include thefollowing factors: 1) the indicator should always be present in thepresence of pathogens; 2) the indicator should always be present in apredicable ratio with pathogenic organisms; 3) the indicator should bespecific for fecal contamination; 4) the indicator should be able toresist water treatment and disinfection processes to the same or aslightly greater extent than the pathogens; and 5) the indicator shouldbe detectable by simple and rapid methods.

Historically, "coliforms" have served as the indicator bacteria forfecal contamination in United States water supplies. However, term"coliform" encompasses four genera (Escherichia, Citrobacter,Enterobacter, and Klebsiella); many of these species are commonly foundin the environment in the absence of fecal contamination. Although allof these genera may be recovered from domestic sewage in large numbers,only E. coli is consistently and exclusively found in feces (see e.g.,A. P. Dufour, "E. coli: the fecal coliform, in A. W. Hoadley and B. J.Dutka, Bacterial Indicators/Health Hazards Associated with Water, [ASTM,Philadelphia, 1976], p. 48). Thus, coliform detections methods are notspecific for the determination of whether a water supply has beencontaminated with fecal matter. Nonetheless, regulations based ondetection and enumeration of "total coliforms" have been in effect inthe United States since 1914 (i.e., the Treasury Department Standards of1914; subsequent standards have been promulgated by the U.S. PublicHealth Service, and presently, by the US. Environmental ProtectionAgency [EPA]).

Recognition of the fact that most of the organisms included in thedesignation "total coliforms" are not of fecal origin, led to thedevelopment of tests to detect "fecal coliforms," for a subgroup ofthermotolerant organisms included within the total coliforms. However,this designation is also not specific, as it includes E. coli, as wellas various Klebsiella strains. Despite the fact that although there aresubstantial extra-fecal sources of Klebsiella, and this organism isinfrequently found in human feces, the use of the "fecal coliform"designation and tests to identify these organisms remain routine(reviewed by V. J. Cabelli, Health Effects Criteria for MarineRecreational Waters, EPA-600/1-80-031, [August, 1983], pp.11-12).

Furthermore, the correlation between coliform densities in water and theincidence of waterbome disease originally postulated by Kehr andButterfield in 1943 (R. W. Kehr and C. T. Butterfield, "Notes on therelationship between coliforms and enteric pathogens," Public HealthRepts. 58:589-596 [1943]) have not been supported by experimental tests(Batik et al., "Routine monitoring and waterborne disease outbreaks," J.Environ. Health 45:227-230 [1984]). Quite simply, there has been nodirect evidence presented that the level of coliform contaminationcorrelates well with waterborne disease outbreaks (see Pipes, p.434-435). Nonetheless, due to the lack of better methods, the detectionof coliforms as indicator bacteria continues into the present.

Coliform detection may be accomplished by various methods, includingmultiple tube fermentation (i.e., most probable number or "MPN"determinations), membrane filtration, the "presence-absence" test, andvarious rapid enzyme (e.g., the MUG test) and immunoassay methods.Important considerations with these methods include the large time,equipment and personnel commitment necessary to conduct and interpretthese tests.

COLIFORM DETECTION METHODS

Most Probable Number (MPN). The MPN method is a labor, time and supplyintensive method, which involves three distinct stages of specimenprocessing (the presumptive (with lauryl tryptose broth), completed(with brilliant green lactose bile broth) and confirmed tests (with LESEndo or EMB). FIG. 1 illustrates the steps involved in MPN analysis fordetection of coliforms. As is apparent from this figure, the MPN methodrequires 3-4 days in order to produce confirmatory results, andstatistical analysis to quantitate the organisms present.

This procedure has been developed to separate organisms within thecoliform group into "total" and "fecal" coliforms. Prior enrichment oforganisms in a presumptive test medium is required for optimum recoveryof fecal coliforms. These methods are used as confirmatory testsconducted with various selective media and elevated incubationtemperatures (e.g., 44.5° C.). Thus, there is also a significant timeand labor commitment associated with these methods.

Membrane Filtration. In membrane filtration, a known volume of watersample is passed through a membrane filter which is then placed ongrowth media (e.g., M-Endo or LES-Endo), and incubated overnight. Allcolonies with characteristics common to coliforms are considered to bemembers of the coliform group. An advantage of membrane filtration isthat preliminary results are usually available in 24 hours. However,verification of colony identification is recommended, usually requiringadditional days in order to conduct the needed biochemical tests.

Additional disadvantages with the membrane filtration method includefouling of membranes with debris and suspended solids present in water.These particulates prevent free flow of water through the membrane,greatly slowing the process. In addition, the presence of particulatematerial on the membrane often interferes with organism growth,preventing reliable identification of bacteria. In this situation,reliable enumeration estimates are also precluded due to the presence ofvisible particulates present on the membrane which may be confused withcolonies, the possibility that colonies are present under theparticulate matter, yet not be visible for counting, and the potentialinterference with organism growth due to the composition of theparticulates (e.g., the particulate may be comprised of a material toxicto the organisms).

Membrane filtration methods are especially unsuitable for use with"dirty" water. This is a significant consideration in many settings,especially testing of environmental waters.

Membrane Filtration Method Modifications. A seven hour fecal coliformtest similar to the membrane filtration process has also been described.In this technique, the water sample is filtered and the filter placed onM-7 FC agar and incubated at 41.5° C. [American Public HealthAssociation-American Water Works Association-Water Pollution ControlFederation, Standard Methods for the Examination of Water andWastewater, 16th ed., [APHA, Washington, D.C.], 1985; hereinafter,"Standard Methods"]. Yellow colonies representing fecal coliforms areenumerated after seven hours of incubation. However, different growthrates of colonies necessitate a compromise between sensitivity ofdetection and enumeration. That is to say, because different organismsgrow at different rates, some organisms will not have had sufficienttime to produce visible colonies on the medium by the time enumerationis conducted.

The value of this test is perhaps questionable, in view of its deletionfrom the most recent edition of Standard Methods.

Another method developed by Reasoner, in conjunction with Geldrich [D.J. Reasoner and E. E. Geldreich, "Rapid detection of water-borne fecalcoliforms by ¹⁴ CO₂ release," in A. N. Sharpe and D. S. Clark, (eds.)Mechanizing Microbiology, [Charles C. Thomas Publishers, 1978], pp.120-139) involves concentration of bacteria on a membrane filter whichis then placed in M-FC broth which contains radiolabelled ¹⁴ C-mannitol.Major problems with these methods involve the use of radioactivity andthe attendant disposal and handling concerns, as well as the need forspecialized and expensive instruments. The tubes are incubated for 2hours at 35° C., followed by 2.5 hours at 44.5°. Release of ¹⁴ CO₂ dueto microbial metabolism is then assayed by liquid scintillationspectrometry.

An alternate radioactive test was developed by Dange et al. (V. Dange etal., "One hour portable test for drinking waters," Water Res.,22:133-137 [1988]). This method is based on the correlation of ³² Puptake by organisms present in a water sample incubated in a syntheticmedium. Thus, these methods require highly trained laboratory personneland are not suitable for use in many labs.

Presence-Absence Test. The presence-absence test to detect the presenceof coliforms involves the inoculation of broth with 100 ml samples ofwater, followed by incubation at 25° C. for 24-48 hours. If acid and gasis produced in the medium, the test is positive for the presence ofcoliforms (see e.g., Standard Methods, at p. 882-884). No enumeration oforganisms is attempted, nor are any identification methods utilized.Thus, the information garnered from this method is very limited.

Fluorometric and Enzymatic Tests. Detection methods for coliforms withfluorometric tests and numerous variations on the basic technology havealso been developed. Other substrate-based methods include the use ofsuch compounds as ortho-nitrophenyl-β-D-galactopyranoside (ONPG) and4-methylumbelliferyl-β-D-glucuronide (MUG). These methods utilizefluorogenic or chromogenic substrates to detect coliform metabolism, asopposed to direct detection and enumeration of organisms. Thus, the onlydata available from these test methods relate to the presence or absenceof organisms which possess the necessary enzymatic machinery to producethe detectable color compounds from a given substrate.

The MUG test is also problematic in that many clinically important E.coli strains are negative. For example, the highly virulent and verydifficult to treat, E. coli 0157:H7 serotype associated with recentfoodborne disease outbreaks is negative in this test (see e.g., E. W.Frampton and L. Restaino, "Methods for Escherichia coli identificationin food, water and clinical samples based on beta-glucuronidasedetection," J. Appl. Bacteriol., 74:223-233 [1993]). Indeed, there is alarge proportion of β-glucuronidase negative E. coli (see e.g., G. W.Chang et al., "Proportion of β-D-glucuronidase-negative Escherichia coliin human fecal samples," Appl. Environ. Microbiol., 55:335-339 [1989]).Furthermore, species within other genera such as Staphylococcus,Streptococcus, Clostridium, and the anaerobic corynebacteria alsoproduce β-glucuronidase (Frampton and Restaino, p. 223). Thus, not onlyis the test not highly sensitive, it is not specific. These reportsraise serious questions regarding the reliability of these testingmethods.

Bacteriophages. In addition to culture and enzymatic detection methods,bacteriophages have also been used with some limited success asindicators of fecal contamination (R. S. Wensel et al., "Evaluation ofcoliphage detection as a rapid indicator of water quality," Appl.Environ. Microbiol., 43:430-434 [1982]; Y. Kott et al., "Bacteriophagesas bacterial viral pollution indicators," Water Res., 8:165-171 [1982];and A. H. Havelaar et al., "Factors effecting the enumeration ofcoliphages in sewage and sewage-polluted waters," Antonie van Leewenhoek49:387-397 [1983]).

However, the detection limits provided by these methods are no betterthan those obtained with standard methods for water qualitydeterminations based on coliform analysis. Thus, these methods do notprovide a significant advantage over the traditional methods of wateranalysis.

In summary, the coliform group falls far short of the ideal indicatorsystem. Coliform-free drinking water has been implicated in severalwaterbome outbreaks [see e.g., B. J. Dutka, "Coliforms are inadequateindex of water quality," J. Environ. Health 36:39-46 [1973]). Likewise,the presence of coliforms in a particular water sample does notnecessarily correlate well with the incidence of disease.

Even the enumeration of "fecal coliforms" is less than optimal, as someorganisms such as Klebsiella are capable of producing positive testresults. Such observations led to the development of alternativeindicator organisms, including tests specific for E. coli, fecalstreptococci (e.g., enterococci), Klebsiella, Clostridium perfringens,Pseudomonas aeruginosa, Bifidobacterium, Bacteroides, Aeromonashydrophila, V. parahaemolyticus, and C. albicans, as well as otherorganisms commonly excreted in large numbers by healthy mammals.Notably, various opportunistic and frank pathogens uncommonly associatedwith waterborne transmission of disease are included in the list ofindicator organisms (e.g., C. perfringens, A. hydrophila, V.parahaemolyticus, and C. albicans. Although these organisms may beuseful in some settings as predictors of waterborne disease, whatremains to be developed is a method for the detection and enumeration ofpathogens commonly associated with waterborne diarrheal illness.

Detection and identification of Salmonella and Shigella from clinicalsamples has traditionally involved microbiological cultures, biochemicalanalyses and in some cases, serotyping methods. The same methods areused to identify suspected Salmonella or Shigella colonies isolated fromclinical samples are also usually used for water, food, and otherenvironmental samples. However, these methods are not well-suited to theunique situations associated with environmental samples, where many ofthe organisms present are stressed and do not perform as expected inclinical testing methods. For example, in the case of Shigella, testingproblems arise due to the instability of some biochemicalcharacteristics and antagonism of E. coli and Proteus vulgaris towardShigella (Standard Methods, p. 927).

Despite years of regulation and testing, development of a bacterialindicator which is directly related to fecal contamination and/or thepresence of pathogens which can cause waterborne disease (Pipes, p. 449)is desirable. Thus, what is needed is a cost-effective method, which isat least as sensitive and specific as traditional methods for the directdetection of pathogens present in clinical, food, water and otherenvironmental samples.

SUMMARY OF THE INVENTION

In one embodiment, the present invention provides a method foramplification of nucleic acid from one or more enteric pathogenscomprising the steps of: a) providing a test sample suspected ofcontaining amplifiable nucleic acid one or more one enteric pathogens;b) isolating the amplifiable nucleic acid from the test sample; c)combining the amplifiable nucleic acid with amplification reagents, andat least two primers selected from the group consisting of primershaving the nucleic acid sequence set forth in SEQ ID NO:1, SEQ ID NO:2,SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8 and SEQID NO:9, to form a reaction mixture; and d) combining the reactionmixture with an amplification enzyme under conditions wherein theamplifiable nucleic acid is amplified to form amplification product. Itis particularly contemplated that SEQ ID NOS: 1 and 2 may be combinedwith SEQ ID NOS: 3 and 4 in order to amplify Salmonella sequences. It isalso particularly contemplated that SEQ ID NOS: 6 and 7 may be combinedwith SEQ ID NOS:8 and 9 in order to amplify Shigella

In a preferred embodiment, the method of the present invention furthercomprises the step of detecting the amplification product. It iscontemplated that this detection will be accomplished by hybridizationof the amplification product with a probe having the nucleic acidsequence set forth in SEQ ID NO:5. It is also contemplated that thisdetection will be accomplished by hybridization of the amplificationproduct with a probe having the nucleic acid sequence set forth in SEQID NO:8 or SEQ ID NO:9.

In one embodiment of the present invention, the method utilizes primerscapable of hybridizing to nucleic acid from the enteric pathogensSalmonella and Shigella. It is further contemplated that any Salmonellaor Shigella species, subspecies, strain or type will be detected by themethod of the present invention.

In one embodiment, the test sample is selected from the group comprisingsewage, sludge, soil, food, feed, and water. It is thereforecontemplated that the test sample comprise any number of sample types,both environmental and clinical.

In an alternative embodiment, the present invention comprises a methodfor amplification of nucleic acid from a plurality of enteric pathogenscomprising the steps of: a) providing a test sample suspected ofcontaining amplifiable nucleic acid of a plurality of enteric pathogens;b) isolating the amplifiable nucleic acid from the test sample; c)combining the amplifiable nucleic acid with amplification reagents, andall of the primers set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:6and SEQ ID NO:7, to form a reaction mixture; d) combining the reactionmixture with an amplification enzyme under conditions wherein theamplifiable nucleic acid is amplified to form amplification products;and e) detecting the amplification products.

In a preferred embodiment of this method the detecting compriseshybridizing the amplification products with the probes having thenucleic acid sequences set forth in SEQ ID NO:5, and SEQ ID NO:8 or SEQID NO:9. In another embodiment, the combining conditions comprise atemperature range of 57° C. to 58° C.

In another preferred embodiment, the enteric pathogens detected by thismethod are Salmonella and Shigella. In one embodiment, the test sampleis selected from the group comprising sewage, sludge, soil, food, feed,and water.

The present invention also contemplates a method for amplification ofnucleic acid from an enteric pathogen comprising the steps of: a)providing a test sample suspected of containing amplifiable nucleic acidof an enteric pathogens; b) isolating said amplifiable nucleic acid fromthe test sample; c) combining the amplifiable nucleic acid withamplification reagents, and the primers set forth in SEQ ID NO:1, SEQ IDNO:2, SEQ ID NO:3, and SEQ ID NO:4, to form a reaction mixture; d)combining the reaction mixture with an amplification enzyme underconditions wherein the amplifiable nucleic acid is amplified to formamplification product; and e) detecting said amplification product. Inthis embodiment, it is contemplated that a the first reaction mixturewill be produced with primers of SEQ ID NOS: 1 and 2. It is thencontemplated that a portion or aliquot of this first reaction mixturewill be again subjected to PCR using the primers of SEQ ID NOS: 3 and 4in order to produce a second reaction mixture. It is contemplated thatonly a portion of this first reaction mixture will be necessary for usein the PCR process with the second set of primers. This use of a"portion" of the first reaction mixture provides advantages in that asmaller initial sample volume may be used, as a second round ofamplification is involved.

In a preferred embodiment of this method, the detecting compriseshybridizing the amplification product with a probe having the nucleicacid sequence set forth in SEQ ID NO:5. It is particularly contemplatedthat this probe is labelled. For example, the probe may be labelled witha radioactive or fluorescent compound or any other reporter molecule. Ina particularly beneficial embodiment, the conditions comprise atemperature range of 57° C. to 58° C. In another preferred embodiment,the enteric pathogen is Salmonella.

In one embodiment of this invention, the test sample is selected fromthe group comprising sewage, sludge, soil, food, feed, and water.

In an additional embodiment, present invention also contemplates amethod for amplification of nucleic acid from an enteric pathogencomprising the steps of: a) providing a test sample suspected ofcontaining amplifiable nucleic acid of an enteric pathogens; b)isolating said amplifiable nucleic acid from the test sample; c)combining the amplifiable nucleic acid with amplification reagents, andthe primers set forth in SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, and SEQID NO:9, to form a reaction mixture; d) combining the reaction mixturewith an amplification enzyme under conditions wherein the amplifiablenucleic acid is amplified to form amplification product; and e)detecting said amplification product. In this embodiment, it iscontemplated that a the first reaction mixture will be produced withprimers of SEQ ID NOS: 6 and 7. It is then contemplated that a portionor aliquot of this first reaction mixture will be again subjected to PCRusing the primers of SEQ ID NOS: 8 and 9 in order to produce a secondreaction mixture. It is contemplated that only a portion of this firstreaction mixture will be necessary for use in the PCR process with thesecond set of primers. This use of a "portion" of the first reactionmixture provides advantages in that a smaller initial sample volume maybe used, as a second round of amplification is involved.

In a preferred embodiment of this method, the detecting compriseshybridizing the amplification product with a probe having the nucleicacid sequence set forth in SEQ ID NO:8 or SEQ ID NO:9. It isparticularly contemplated that this probe is labelled. For example, theprobe is labelled with a radioactive or fluorescent compound or anyother reporter molecule. In this embodiment, the probe may be of thesame sequence as one of the primers. In a particularly beneficialembodiment, the conditions comprise a temperature range of 57° C. to 58°C. In another preferred embodiment, the enteric pathogen is Shigella.

The present invention also contemplates a composition comprising one ormore isolated and purified nucleic acid sequences selected from thegroup consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4,SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9.

In a preferred embodiment, the composition further comprises anamplifiable nucleic acid of one or more one enteric pathogens, to form areaction mixture. In one variation of this embodiment, the entericpathogen is Salmonella. In another variation, the enteric pathogen isShigella. In yet another embodiment, there is a combination of entericpathogens, in particular Salmonella and Shigella. It is contemplatedthat this composition will be of particular value in the detection ofthese enteric pathogens in such environmental samples as water andsewage.

DESCRIPTION OF THE DRAWINGS

FIG. 1 is a flow chart of the MPN procedure.

FIG. 2 is a flow chart for isolation of Salmonella from environmentalwater.

FIG. 3 is a gel showing amplification of Salmonella and E. coli.

FIG. 4 is a gel showing amplification of Salmonella and E. coli.

FIG. 5 is a gel showing amplification of Salmonella and E. coli.

FIG. 6 is a gel showing amplification of Salmonella and E. coli.

FIG. 7 is a gel showing amplification of Salmonella and E. coli.

FIG. 8 is a gel showing amplification of Salmonella and E. coli.

FIG. 9 is a gel showing detection of Salmonella in seeded sewage.

FIG. 10 is a gel showing detection of Salmonella in environmental water.

FIG. 11 is a gel showing PCR products obtained using nested PCR withSalmonella.

FIG. 12 is a gel showing PCR products obtained with Shigella.

FIG. 13 is a gel showing PCR products obtained during simultaneousamplification of Salmonella and Shigella.

DESCRIPTION OF THE INVENTION

The method of the present invention comprises a method for the directdetection of enteric pathogens such as Salmonella and Shigella inenvironmental and food samples. Encompassed within this invention aremethods for processing samples, including particular assay conditionsand/or reagents. Importantly, the reagents and method of the presentinvention obviate the traditional methods of water quality analysisusing indicator bacteria.

In a preferred embodiment, a pair of DNA primers, each 20 base pairs inlength is used for the detection of Salmonella in a polymerase chainreaction (PCR) method. In an alternate preferred embodiment, a secondpair of primers (19 base pairs each) is used in a nested PCR assay. Anadditional set of primers (19 base pairs each) was developed fordetection of Shigella. The combined use of these primers permits therapid and specific detection of Salmonella and Shigella.

Importantly, the invention permits the rapid detection of as few as 10Salmonella organisms in less than five (5) hours. This detection methodis both highly sensitive and specific. Importantly, the method of thepresent invention does not require a pre-culturing step prior to PCR.Thus, the method and reagents of the present invention permit rapiddiagnosis of Salmonella and Shigella infection, a concern of greatimportance in potentially life-threatening diseases (e.g., typhoid) andepidemiologic investigations.

This is also significant for detection of pathogens in water and othersamples, especially for organisms such as Shigella with low infectivedoses. Estimates indicate that as few as 200 organisms is all that isrequired to produce disease in healthy adults (P. Zwadyk,"Enterobacteriaceae: Salmonella and Shigella, intestinal pathogens," pp.613-622 in W. K. Joklik et al., (eds), Zinsser Microbiology, 18thedition [Appleton Century Crofts, Norwalk, Conn., 1984]). Thus, exposureto a very few organisms may result in disease. Given the incidence ofshigellosis (or "bacillary dysentery"), with approximately 500,000deaths reported annually (see e.g., Hale, p. 206), detection of thisorganism is highly desirable.

The rapid, sensitive and specific detection method of the presentinvention is therefore extremely well-suited for the detection of lownumbers of pathogens. Thus, it is contemplated that the presentinvention be used in various settings, including, but not limited to,such areas as clinical, veterinary, food, dairy, and feed industries,and water quality (e.g., drinking water, as well as sewage treatment).

While some previous reports indicate that molecular methods are usefulfor the detection of such pathogens as Salmonella and Shigella, nonedisclose the method nor primers of the present invention. The cumbersomeand error-prone methods described by Steffan and Atlas to enhance PCRefficiency with environmental samples are not necessary with the presentinvention. (R. J. Steffan and R. M. Atlas, "Polymerase chain reaction:Applications in environmental microbiology," Ann. Rev. Microbiol.,45:137-161 [1991].) For example, there is no need to dilute the sampleafter the first few cycles of PCR, nor is there the need to dilute thesample after a completed reaction sequence and then perform anadditional round of amplification (see Steffan and Atlas p. 141 for adescription of this procedure). Nor is there the requirement that theprimer concentration be changed during the PCR cycling, as recommendedby some authors (see Steffan and Atlas, p. 141 for a description).

Two publications describe the same 1.8 kb HindIII fragment from thechromosomal DNA of Salmonella typhimurium, portions of which were usedto develop probes for the detection of Salmonella in foods and othersamples (Tsen et al., "DNA sequence of a Salmonella-specific DNAfragment and the use of oligonucleotide probes for Salmonelladetection," Appl. Microbiol. Biotechnol., 35:339-347 [1991], and Tsen etal., "Possible use of a 1.8 kb DNA fragment for the specific detectionof Salmonella in foods," J. Ferment. Bioeng. 68:1-6 [1989]). Unlike thepresent invention, Tsen et al. did not utilize PCR. Rather, theirdetection methods were based solely on nucleic acid hybridization (i.e.,probe) technology.

Interestingly, while Tsen examined the specificity of six probes havingsequences within this 1.8 kb fragment, only three were found to be"highly specific" for Salmonella. The other three probes (TS1, TS2 andTS3) were found unsuitable because false positive reactions weresometimes observed with E. coli and Citrobacter. This cross-reactivitywas not observed with the present invention.

Definitions

The terms "sample" and "specimen" in the present specification andclaims are used in their broadest sense. These terms are also usedinterchangeably. On the one hand they are meant to include a specimen orculture. On the other hand, they are meant to include both biologicaland environmental samples. In addition, a "sample" may or may notcontain nucleic acid. Furthermore, it may or may not contain "sampletemplate" and/or "background template."

Biological samples may be animal, including human, fluid or tissue, foodproducts and ingredients such as dairy items, vegetables, meat and meatby-products, and waste. Environmental samples include environmentalmaterial such as surface matter, soil, water, wastewater, sewage,sludge, industrial samples (e.g., industrial water), as well as samplesobtained from food and dairy processing instruments, apparatus,equipment, disposable and non-disposable items. Also included aresamples obtained from animals which live in fresh, brackish and/orseawater (e.g., shellfish, fish, and marine animals). In addition tothese "environmental" samples, it is contemplated that "drinking water"will be used with the method of the present invention. It is intendedthat the term "drinking water" encompass all types of water used forconsumption by humans and other animals, including but not limited towell water, run-off water, water stored in reservoirs, rivers, streams,etc. These examples are not to be construed as limiting the sample typesapplicable to the present invention.

The terms also encompass all types of samples obtained from humans andother animals, including but not limited to, body fluids such as urine,blood, fecal matter, and cerebrospinal fluid (CSF), as well as solidtissue. Also included are swabs and other sampling devices which arecommonly used to obtain samples for culture of microorganisms. Thesehuman and veterinary samples are included within the term "clinical"samples.

Whether biological or environmental, a sample suspected of containingmicroorganisms may or may not first be subjected to an enrichment meansto create a "pure culture" of microorganisms. By "enrichment means" or"enrichment treatment," the present invention contemplates (i)conventional techniques for isolating a particular microorganism ofinterest away from other microorganisms by means of liquid, solid,semi-solid or any other culture medium and/or technique, and (ii) noveltechniques for isolating particular microorganisms away from othermicroorganisms. It is not intended that the present invention be limitedonly to one enrichment step or type of enrichment means. For example, itis within the scope of the present invention to, following subjecting asample to a conventional enrichment means, subjecting the resultantpreparation to further purification such that pure or substantially purecultures of a strain of a species of interest are produced.

As used herein, the term "organism" is used to refer to any species ortype of microorganism, including but not limited to bacteria, viruses,fungi, and protozoans.

As used herein, the term "culture" refers to any sample or specimenwhich is suspected of containing one or more microorganisms. "Purecultures" are cultures in which the organisms present are only of onegenus and species. "Mixed cultures" are cultures in which more than onegenus and/or species of microorganism are present.

As used herein, the terms "microbiological media" and "culture media,"and "media" refer to any substrate for the growth and reproduction ofmicroorganisms. "Media" may be used in reference to solid plated mediawhich support the growth of microorganisms. Also included within thisdefinition are microbial growth systems which incorporate living hostorganisms, as well as any type of media. Such systems include, but arenot limited to the cell culture systems utilized to grow variousfastidious organisms

As used herein, the term "selective media" refers to media which supportthe growth of particular organisms of interest but inhibit otherorganisms. Such inhibition may result due to medium constituents such ascompounds which are selectively toxic, as well as the end-products ofmicrobial metabolism produced by organisms which utilize the mediumconstituents.

As used herein, the term "differential media" refers to media whichsupport the growth of various organisms, but permit visualdifferentiation between the different genera or species.

As used herein, the term "primary isolation" refers to the process ofculturing organisms directly from the sample. Thus, primary isolationinvolves such processes as inoculating an agar plate from a cultureswab, water sample, etc. Primary isolation may also be done in liquid orsemi-solid media.

As used herein, the term "presumptive diagnosis" refers to a preliminarydiagnosis which gives some guidance to the treating physician as to theetiologic organism involved in the patient's disease. Presumptivediagnoses are often based on "presumptive identifications," which asused herein refer to the preliminary identification of a microorganismbased on observation such as colony characteristics, growth on primaryisolation media, gram stain results, etc.

As used herein, the term "definitive diagnosis" is used to refer to afinal diagnosis in which the etiologic agent of the patient's diseasehas been identified. As used herein, this term is also applicable to theidentification of organisms present in environmental samples.

As used herein, the term "base" refers to a monomeric unit of nucleicacid. Technically, the monomeric units of DNA are termed"deoxyribonucleotides" and those of RNA are "ribonucleotides." Eachnucleotides is comprised of: 1) a nitrogenous heterocyclic base, 2) apentose, and 3) a molecule of phosphoric acid. Since the nucleotide isdistinguished by the type of base, a shorthand reference for nucleotideshas evolved--the nucleotide is simply referred to as a "base."

As used herein, the terms "complementary" or "complementarity" are usedin reference to polynucleotides (i.e., a sequence of nucleotides)related by the base-pairing rules. For example, for the sequence"A-G-T," is complementary to the sequence "T-C-A." Complementarity maybe "partial," in which only some of the nucleic acids' bases are matchedaccording to the base pairing rules. Or, there may be "complete" or"total" complementarity between the nucleic acids. The degree ofcomplementarity between nucleic acid strands has significant effects onthe efficiency and strength of hybridization between nucleic acidstrands. This is of particular importance in amplification reactions, aswell as detection methods which depend upon binding between nucleicacids.

As used herein, the term "hybridization" is used in reference to thepairing of complementary nucleic acids. Hybridization and the strengthof hybridization (i.e., the strength of the association between thenucleic acids) is impacted by such factors as the degree ofcomplementary between the nucleic acids, stringency of the conditionsinvolved, the T_(m) of the formed hybrid, and the G:C ratio within thenucleic acids.

As used herein, the term "amplifiable nucleic acid" is used in referenceto nucleic acids which may be amplified by any amplification method. Itis contemplated that "amplifiable nucleic acid" will usually comprise"sample template."

As used herein, the term "sample template" refers to nucleic acidoriginating from a sample which is analyzed for the presence of "target"(defined below). In contrast, "background template" is used in referenceto nucleic acid other than sample template which may or may not bepresent in a sample. Background template is most often inadvertent. Itmay be the result of carryover, or it may be due to the presence ofnucleic acid contaminants sought to be purified away from the sample.For example, nucleic acids from organisms other than those to bedetected may be present as background in a test sample.

As used herein, the term "primer" refers to an oligonucleotide, whetheroccurring naturally as in a purified restriction digest or producedsynthetically, which is capable of acting as a point of initiation ofsynthesis when placed under conditions in which synthesis of a primerextension product which is complementary to a nucleic acid strand isinduced, (i.e., in the presence of nucleotides and an inducing agentsuch as DNA polymerase and at a suitable temperature and pH). The primeris preferably single stranded for maximum efficiency in amplification,but may alternatively be double stranded. If double stranded, the primeris first treated to separate its strands before being used to prepareextension products. Preferably, the primer is anoligodeoxyribonucleotide. The primer must be sufficiently long to primethe synthesis of extension products in the presence of the inducingagent. The exact lengths of the primers will depend on many factors,including temperature, source of primer and the use of the method.

As used herein, the term "probe" refers to an oligonucleotide (i.e., asequence of nucleotides), whether occurring naturally as in a purifiedrestriction digest or produced synthetically, which is capable ofhybridizing to another oligonucleotide of interest. Probes are useful inthe detection, identification and isolation of particular genesequences. It is contemplated that any probe used in the presentinvention will be labelled with any "reporter molecule," so that isdetectable in any detection system, including, but not limited to enzyme(e.g., ELISA as well as enzyme-based histochemical assays), fluorescent,radioactive, and luminescent systems. It is further contemplated thatthe oligonucleotide of interest (i.e., to be detected) will be labelledwith a reporter molecule. It is also contemplated that both the probeand oligonucleotide of interest will be labelled. It is not intendedthat the present invention be limited to any particular detection systemor label.

As used herein, the term "target" refers to the region of nucleic acidbounded by the primers used for polymerase chain reaction. Thus, the"target" is sought to be sorted out from other nucleic acid sequences. A"segment" is defined as a region of nucleic acid within the targetsequence.

As used herein, and incorporated by reference, the term "polymerasechain reaction" ("PCR") refers to the method of K. B. Mullis U.S. Pat.Nos. 4,683,195 and 4,683,202, which describe a method for increasing theconcentration of a segment of a target sequence in a mixture of genomicDNA without cloning or purification. This process for amplifying thetarget sequence consists of introducing a large excess of twooligonucleotide primers to the DNA mixture containing the desired targetsequence, followed by a precise sequence of thermal cycling in thepresence of a DNA polymerase. The two primers are complementary to theirrespective strands of the double stranded target sequence. To effectamplification, the mixture is denatured and the primers then annealed totheir complementary sequences within the target molecule. Followingannealing, the primers are extended with a polymerase so as to form anew pair of complementary strands. The steps of denaturation, primerannealing and polymerase extension can be repeated many times (i.e.,denaturation, annealing and extension constitute one "cycle"; there canbe numerous "cycles") to obtain a high concentration of an amplifiedsegment of the desired target sequence. The length of the amplifiedsegment of the desired target sequence is determined by the relativepositions of the primers with respect to each other, and therefore, thislength is a controllable parameter. By virtue of the repeating aspect ofthe process, the method is referred to as the "polymerase chainreaction" (hereinafter "PCR"). Because the desired amplified segments ofthe target sequence become the predominant sequences (in terms ofconcentration) in the mixture, they are said to be "PCR amplified".

With PCR, it is possible to amplify a single copy of a specific targetsequence in genomic DNA to a level detectable by several differentmethodologies (e.g., hybridization with a labeled probe; incorporationof biotinylated primers followed by avidin-enzyme conjugate detection;incorporation of ³² P-labeled deoxynucleotide triphosphates, such asdCTP or dATP, into the amplified segment). In addition to genomic DNA,any oligonucleotide sequence can be amplified with the appropriate setof primer molecules. In particular, the amplified segments created bythe PCR process itself are, themselves, efficient templates forsubsequent PCR amplifications.

"Amplification" is a special case of nucleic acid replication involvingtemplate specificity. It is to be contrasted with non-specific templatereplication (i.e., replication that is template-dependent but notdependent on a specific template). Template specificity is heredistinguished from fidelity of replication (i.e., synthesis of theproper polynucleotide sequence) and nucleotide (ribo- or deoxyribo-)specificity. Template specificity is frequently described in terms of"target" specificity. Target sequences are "targets" in the sense thatthey are sought to be sorted out from other nucleic acid. Amplificationtechniques have been designed primarily for this sorting out.

Template specificity is achieved in most amplification techniques by thechoice of enzyme. Amplification enzymes are enzymes that, underconditions they are used, will process only specific sequences ofnucleic acid in a heterogenous mixture of nucleic acid. For example, inthe case of Qβ replicase, MDV-1 RNA is the specific template for thereplicase. D. L. Kacian et al., Proc. Nat. Acad. Sci USA 69:3038 (1972).Other nucleic acid will not be replicated by this amplification enzyme.Similarly, in the case of T7 RNA polymerase, this amplification enzymehas a stringent specificity for its own promoters. M. Chamberlin et al.,Nature 228:227 (1970). In the case of T4 DNA ligase, the enzyme will notligate the two oligonucleotides where there is a mismatch between theoligonucleotide substrate and the template at the ligation junction. D.Y. Wu and R. B. Wallace, Genomics 4:560 (1989). Finally, Taq polymerase,by virtue of its ability to function at high temperature, is found todisplay high specificity for the sequences bounded and thus defined bythe primers; the high temperature results in thermodynamic conditionsthat favor primer hybridization with the target sequences and nothybridization with non-target sequences. PCR Technology, H. A. Erlich(ed.) (Stockton Press 1989).

Some amplification techniques take the approach of amplifying and thendetecting target; others detect target and then amplify probe.Regardless of the approach, nucleic acid must be free of inhibitors foramplification to occur at high efficiency.

As used herein, the terms "PCR product" and "amplification product"refer to the resultant mixture of compounds after two or more cycles ofthe PCR steps of denaturation, annealing and extension are complete.These terms encompass the case where there has been amplification of oneor more segments of one or more target sequences.

As used herein, the term "nested primers" refers to primers that annealto the target sequence in an area that is inside the annealingboundaries used to start PCR. K. B. Mullis, et al., Cold Spring HarborSymposia, Vol. II, pp. 263-273 (1986). Because the nested primers annealto the target inside the annealing boundaries of the starting primers,the predominant PCR-amplified product of the starting primers isnecessarily a longer sequence, than that defined by the annealingboundaries of the nested primers. The PCR-amplified product of thenested primers is an amplified segment of the target sequence thatcannot, therefore, anneal with the starting primers. Advantages to theuse of nested primers include the large degree of specificity, as wellas the fact that a smaller sample portion may be used and yet obtainspecific and efficient amplification.

As used herein, the term "multiplex" is used in reference toamplification of multiple nucleic acid sequences (e.g., PCR procedureswhich include multiple primer sets to simultaneous amplify nucleic acidsfrom multiple sources). In multiplex PCR, specific, sensitive anddistinguishable simultaneous amplification of gene sequences fromdifferent organisms (e.g., Salmonella and Shigella) may be conducted.Because more than one organism may be detected and identified inmultiplex reactions, this procedure may provide significant advantagesover single PCR methods in which only one gene sequence is detected.

As used herein, the term "amplification reagents" refers to thosereagents (deoxyribonucleoside triphosphates, buffer, etc.), needed foramplification except for primers, nucleic acid template and theamplification enzyme. Typically, amplification reagents along with otherreaction components are placed and contained in a reaction vessel (testtube, microwell, etc.).

As used herein, the terms "restriction endonucleases" and "restrictionenzymes" refer to bacterial enzymes, each of which cut double-strandedDNA at or near a specific nucleotide sequence.

As used herein, the terms "polymerase inhibitors," and "interfering"compounds or "substances" include all compounds (organic and inorganic)that reduce the amount of nucleic acid replication by enzymes. It is notintended to be limited by the mechanism by which these inhibitors orinterfering compounds achieve this reduction. Furthermore, it is notintended to be limited to only those inhibitors which display largereductions.

It is contemplated that methods to avoid problems associated with"carryover" will by used in conjunction with the present invention. Asused herein, the term "carryover" is broadly defined as nucleic acidthat is accidentally introduced into a reaction mixture. Of course, thetypes of accidental introductions are numerous. Nucleic acids can beintroduced during a spill or because of poor laboratory technique (e.g.,using the same reaction vessel or the same pipette twice). However, ofgreater concern is the introduction of nucleic acids that occurs evenduring normal laboratory procedures, including inadvertent transfer fromcontaminated gloves.

It is contemplated that any of the various approaches to controllingcarryover reported in the literature will be used with the presentinvention, including containment, elimination, prevention, and/or"sterilization" (both post-amplification sterilization, as well aspre-amplification sterilization). For example, the "sterilization"methods as those described in U.S. Pat. No. 5,139,940 issued to Isaacset al., and U.S. Pat. No. 5,221,608 issued to Cimino et al., are herebyincorporated by reference and may be used with the present invention.

As used herein, the term "T_(m) " is used in reference to the "meltingtemperature." The melting temperature is the temperature at which apopulation of double-stranded nucleic acid molecules becomes halfdissociated into single strands. The equation for calculating the T_(m)of nucleic acids is well known in the art. As indicated by standardreferences, a simple estimate of the T_(m) value may be calculated bythe equation: T_(m) =81.5+0.41(% G+C), when a nucleic acid is in aqueoussolution at 1M NaCl (see e.g., Anderson and Young, Quantitative FilterHybridisation, in Nucleic Acid Hybridisation (1985). Other referencesinclude more sophisticated computations which take structural as well assequence characteristics into account for the calculation of T_(m).

As used herein the term "stringency" is used in reference to theconditions of temperature, ionic strength, and the presence of othercompounds such as organic solvents, under which nucleic acidhybridizations are conducted. With "high stringency" conditions, nucleicacid base pairing will occur only between nucleic acid fragments thathave a high frequency of complementary base sequences. Thus, conditionsof "weak" or "low" stringency are often required with nucleic acids thatare derived from organisms that are genetically diverse, as thefrequency of complementary sequences is usually less.

As used herein, the term "detection" is used in reference to theobservation of a product of interest. Detection may refer to isolationof organisms on media, or it may refer to visualization or therecognition of the presence of certain nucleic acids. For example, thePCR products produced in the present invention may be "detected."Detection may be achieved through various methods, including the use of"reporter compounds," including radiolabels, fluorescence, luminescenceor enzymatically-labelled compounds. The efficiency of detection islargely dependent upon the "sensitivity" and "specificity" of themethods employed. As used herein, the term "sensitivity" is used inreference to the ability of the method to detect the item or compound ofinterest when it is present (i.e., detection of "true positives"). Asused herein, the term "specificity" is used in reference to the abilityof the method to detect the particular item or compound or interest, butwithout detecting other compounds or items (i.e., not detecting "falsepositives"). Thus, the greater the specificity, fewer "cross-reactions"will be observed. For example, in nucleic acid detection methods, onlythe particular sequence of interest and no other is detected in a methodthat is highly specific. If the method is sensitive, all of thesequences of interest are detected. Thus, it is clear that the optimalmethods of detection will be both highly sensitive and highly specific.

It is also contemplated that various detection formats will be utilizedwith the present invention, including gels of any composition, beads,immobilized reactants, etc. It is not intended that the method of thepresent invention be limited to a particular detection system or format.

EXPERIMENTAL

The following examples are provided in order to demonstrate and furtherillustrate certain preferred embodiments and aspects of the presentinvention and are not to be construed as limiting the scope thereof.

In the experimental disclosure which follows, the followingabbreviations apply: eq (equivalents); M (Molar); μM (micromolar); N(Normal); mol (moles); mmol (millimoles); μmol (micromoles); nmol(nanomoles); g or gm (grams); mg (milligrams); μg (micrograms); ng(nanograms); 1 or L (liters); ml (milliliters); μl (microliters); cm(centimeters); mm (millimeters); μm (micrometers); nm (nanometers); °C.(degrees Centigrade); CFU (colony forming-units); SDS (sodium dodecylsulfate); PBS (phosphate buffered saline; 137 mM NaCl, 2.7 KCl, 8.1 mMNa₂ HPO₄, 1.5 mM KH₂ PO₄ ; pH 7.3); SSC (0.15M NaCl, 0.015 trisodiumcitrate; pH 7.0); TE buffer (10 mM Tris-HCl, 1.0 mM EDTA; pH 8.0); PCRbuffer (an aqueous solution of 10 mM Tris-HCl, 50 mM KCl, and 1.5 mMMgCl₂, pH 8.3); reaction mixture (1× strength PCR buffer, 200 μm of eachdeoxynucleoside triphosphate [i.e., dNTP's--dATP, dTTP, dCTP, and dGTP],0.3 μM of each primer, and 0.5 μl Taq DNA polymerase [Perkin-Elmer] per100 μl solution); ELISA (Enzyme-Linked Immunosorbent Assay); Millipore(Millipore, Bedford, Mass.; Pharmacia (Pharmacia Fine Chemicals,Piscataway, N.J.); BIO 101 (BIO 101 Inc., La Jolla, Calif.); Difco(Difco Laboratories, Detroit, Mich.); Amicon (Amicon, Beverly, Mass.);Applied Biosystems (Applied Biosystems International, Foster City,Calif.); Perkin Elmer (Perkin-Elmer, Norwalk, Conn.); FMC BioProducts(FMC BioProducts, Rocklane, Me.); BioVentures (BioVentures, Inc.,Murfreesboro, Tenn.); UVP (Ultra Violet Products, San Gabriel, Calif.);Amersham (Amersham, Arlington Heights, Ill.); Stratagene (Stratagene, LaJolla, Calif.); Scientific Products (McGraw Park, Ill.); Sigma (SigmaChemical Co., St. Louis, Mo.); ATCC (American Type Culture Collection,Rockville, Md.); Boehringer Mannheim (Boehringer Mannheim, Indianapolis,Ind.); Eastman Kodak (Eastman Kodak, Rochester, N.Y.); DOH (Departmentof Health, Honolulu, Hi.); MPN (most probable number); MF (membranefiltration). A Perkin-Elmer GeneAmp™ PCR system 9600 was used for all ofthe PCR reactions.

The following table lists the species, number of isolates and sources ofthe organisms used in the following examples. The organisms used in thedevelopment of the present invention were obtained from a variety ofsources. Importantly, in addition to type cultures and lab-adaptedstrains, the present invention was developed using environmentalisolates as well. All Salmonella isolates obtained from the HawaiiDepartment of Health were isolated from clinical samples and wereserotyped. All of these isolates were grown in heart infusion (DifcoLaboratories, Detroit, Mich.) for 24 hrs at 37° C.

                  TABLE 3                                                         ______________________________________                                                        # of                                                                          Isolates                                                      Organism        Tested   Source                                               ______________________________________                                        Salmonella typhimurium                                                                        1        ATCC 14028                                           Salmonella typhimurium                                                                        5        DOH, Honolulu, HI.sup.a                              Salmonella bredeney                                                                           1        DOH, Honolulu, HI.sup.a                              Salmonella heidelberg                                                                         1        DOH, Honolulu, HI.sup.a                              Salmonella infantis                                                                           1        DOH, Honolulu, HI.sup.a                              Salmonella sandiego                                                                           1        DOH, Honolulu, HI.sup.a                              Salmonella muenchen                                                                           1        DOH, Honolulu, HI.sup.a                              Salmonella anatum                                                                             2        DOH, Honolulu, HI.sup.a                              Salmonella hadar                                                                              2        DOH, Honolulu, HI.sup.a                              Salmonella agona                                                                              1        DOH, Honolulu, HI.sup.a                              Salmonella tuebingen                                                                          1        DOH, Honolulu, HI.sup.a                              Salmonella cerro                                                                              1        DOH, Honolulu, HI.sup.a                              Salmonella give 1        DOH, Honolulu, HI.sup.a                              Salmonella alachua                                                                            1        DOH, Honolulu, HI.sup.a                              Salmonella welteureden                                                                        12       DOH, Honolulu, HI.sup.a                              Salmonella newport                                                                            4        DOH, Honolulu, HI.sup.a                              Salmonella lanka                                                                              3        DOH, Honolulu, HI.sup.a                              Salmonella olso 7        DOH, Honolulu, HI.sup.a                              Salmonella singapore                                                                          1        DOH, Honolulu, HI.sup.a                              Salmonella nottingham                                                                         1        DOH, Honolulu, HI.sup.a                              Salmonella tennessee                                                                          1        DOH, Honolulu, HI.sup.a                              Salmonella montevideo                                                                         1        DOH, Honolulu, HI.sup.a                              Salmonella stanley                                                                            1        DOH, Honolulu, HI.sup.a                              Salmonella houten                                                                             2        DOH, Honolulu, HI.sup.a                              Salmonella derby                                                                              1        DOH, Honolulu, HI.sup.a                              Salmonella cubana                                                                             1        DOH, Honolulu, HI.sup.a                              Salmonella dublin                                                                             1        DOH, Honolulu, HI.sup.a                              Escherichia coli                                                                              1        ATCC 25922                                           Escherichia coli                                                                              1        ATCC 35401                                           Escherichia coli                                                                              1        ATCC 43886                                           Escherichia coli                                                                              1        ATCC 43889                                           Escherichia coli                                                                              1        ATCC 43890                                           Escherichia coli                                                                              1        ATCC 43894                                           Escherichia coli                                                                              1        ATCC 43895                                           Escherichia coli                                                                              1        ATCC 43896                                           Enterobacter aerogenes                                                                        1        ATCC 13048                                           Citrobacter freundii                                                                          1        ATCC 8090                                            Klebsiella pneumoniae                                                                         1        ATCC 13883                                           Shigella flexneri                                                                             1        ATCC 12022                                           Shigella sonnei 1        ATCC 25931                                           Bacillus cereus 1        ATCC 14579                                           Bacillus subtilis                                                                             1        ATCC 6051                                            Pseudomonas aeruginosa                                                                        1        ATCC 27853                                           Staphylococcus aureus                                                                         1        ATCC 25923                                           Streptococcus faecalis                                                                        1        ATCC 29212                                           Acinetobacter calcoaceticus                                                                   1        ATCC 19606                                           Serratia marcescens                                                                           1        ATCC 8100                                            Streptococcus pyogenes                                                                        1        ATCC 19615                                           Enterobacter cloacae                                                                          1        ATCC 23355                                           Proteus vulgaris                                                                              1        ATCC 13315                                           Staphylococcus epidermidis                                                                    1        ATCC 12228                                           ______________________________________                                    

EXAMPLE 1 COLLECTION OF ENVIRONMENTAL SAMPLES

Environmental samples were collected in twenty liter volumes from thearea near the Sand Island, Oahu treatment facility's outfall. Thisoutfall is located 9000 ft from shore and is at a depth of 243 ft.Environmental samples were also collected in 20 liter volumes from ManoaStream, a fresh water urban drainage system which serves the Honolulusuburbs.

FIG. 2 is a flow chart illustrating the steps used to isolate Salmonellafrom these samples. Briefly, a 15 tube MPN set up (5 tubes each of 10ml, 1 ml, and 0.1 ml) in tetrathionate broth (Difco)(see e.g., StandardMethods, pp. 880-882, for a description of the procedure) was inoculatedwith either sample water (with no concentration) or water concentratedin a Membrex concentrator, and incubated for 24 hrs at 37° C. Tubesshowing growth after 24 hrs were streaked on xylose lysine deoxycholateagar ("XLD";(Difco) and Hektoen enteric agar ("HE"; Difco) and incubatedfor 24 hrs at 37° C. Characteristic Salmonella-like colonies were theninoculated on triple iron agar ("TSI"; Difco) and lysine iron agar("LIA"; Difco) slants. The identification of isolates with correctgrowth characteristics on TSI and LIA (i.e., for most Salmonellastrains, glucose, but not lactose nor sucrose fermentation, along withgas and H₂ S production in TSI, and lysine decarboxylation in LIAslants) were confirmed with polyvalent antisera specific for SalmonellaO-group antigens (Set A-1, Difco).

EXAMPLE 2 EXTRACTION, PURIFICATION AND CONCENTRATION OF DNA

Mid-log-phase pure cultures were washed twice with phosphate-bufferedsaline (PBS), and DNA extraction was performed using the GNOME DNAisolation kit, (BIO 101), according to the protocol provided by themanufacturer.

Sewage samples were processed using the method of A. K. Bej et al.,"Polymerase chain reaction-gene probe detection of microorganisms byusing filter-concentrated samples," Appl. Environ. Microbiol.,57:3529-3534 (1991). Briefly, this in this protocol, portions (10 ml) ofsamples and dilutions in PBS were filtered through 0.5 μm pore-size FHLPTeflon filters with 13 mm diameters (Millipore) in a Swinnex filterholder (Millipore). This preparation was then used as the source for thetotal DNA, which extracted from the samples by a rapid freeze-thaw. Inthis process, the samples were placed in a dry ice and ethanol bath for1 minute and a 50° C. water bath for 1 minute, and the cycle repeatedfive times.

The samples were then loaded onto a Sephadex G-200 (Pharmacia) columnwhich was then centrifuged twice for 10 mins. each time at 1,100×g toobtain 100 μl of eluent. Approximately 1.5 ml of sterile deionized waterwas added to this final volume. Purified DNA was then concentrated usingan Amicon Centricon concentrator to a final volume of 20 μl. These DNApreparations were then used in the PCR protocols described below.

For environmental water samples, the same filtration process was used asdescribed for sewage. However, rather than the freeze-thaw step, thesesamples were purified by a modification of a previously described method(Y.-L Tsai and B. H. Olson, "Rapid methods for separation of bacterialDNA from humic substances for polymerase chain reaction," Appl. Environ.Microbiol., 58:2292-2295 [1991]; and Y.-L Tsai et al., "Detection ofEscherichia coli in sewage and sludge by polymerase chain reaction,"Appl. Environ. Microbiol., 59:353-357 [1993]). Briefly, 200 μl of thecrude extract was placed in 200 μl of a solution comprising 20% w/vChelex anion exchange resin suspended in 10 mM Tris-HCl, pH 8.0, 1.0 mMEDTA, and 0.1% sodium azide. This suspension was boiled for 10-12 min.,and loaded onto a Sephadex G-200 (Pharmacia) column which was thencentrifuged twice for 10 mins. each time at 1,100×g to obtain 100 μl ofeluent. Purified DNA was then concentrated in the presence of steriledeoinized water, as described above, using an Amicon Centriconconcentrator to a final volume of 20 μl.

Although it was not usually found to be necessary, the Sephadex G-200column was also found to be useful for environmental water with a largeamount of suspended material.

These sewage and environmental water preparations were then used in thePCR protocols described below.

EXAMPLE 3 DEVELOPMENT OF PCR PROTOCOLS

This experiment describes the development and production of the primersutilized in the present invention.

Multiple primers were developed, including primer BR-SALa (SEQ ID NO:1)and BR-SALb (SEQ ID NO:2), which were prepared from a previouslypublished Salmonella restriction fragment sequence (Tsen et al., 1991).A second nested primer set SAL-Ia (SEQ ID NO:3) and SAL-Ib (SEQ ID NO:4)was also prepared from the same large sequence. These primers are shownin Table 4. As shown in Table 5, primer set BR-SALa and BR-SALbgenerated a 526 bp PCR product and nested primer set SAL-Ia and SAL-Ibcorresponded to a 282 bp PCR product. These primers were produced withan automatic DNA synthesizer (ABI 381A; Applied Biosystems).

                  TABLE 4                                                         ______________________________________                                        Primers Used For Detection Of Salmonella                                      Primer   Sequence           SEQ ID NO:                                        ______________________________________                                        BR-SalA  5'-ACG GTT GTT TAG SEQ ID NO: 1                                               CCT GAT AC-3'                                                        BR-SalB  5'-CTG GAT GAG ATG SEQ ID NO: 2                                               GAA GAA TG-3'                                                        BR-Sal IA                                                                              5'-GTT CGG CAT TGT SEQ ID NO: 3                                               TAT TTC T-3'                                                         BR-Sal IB                                                                              5'-CTC AGG GTC ATC SEQ ID NO: 4                                               GTT ATT C-3'                                                         ______________________________________                                    

                  TABLE 5                                                         ______________________________________                                        Position And Products Produced By The Primers                                 Used In The PCR Detection Of Salmonella                                       Primer Set Sequence position.sup.a                                                                     Product length (bp)                                  ______________________________________                                        BR-SALa    1124-1144     526                                                  BR-SALb    1630-1650                                                          SAL-Ia     1280-1299     282                                                  SAL-Ib     1543-1562                                                          ______________________________________                                         .sup.a Position of primers within the 1.8 kb Salmonella DNA sequence (Tse     et al., 1991).                                                           

EXAMPLE 4 HYBRIDIZATION OF PCR PRODUCTS

Following PCR amplification, DNA electrophoresis and hybridizationmethods were used to confirm the sequences of the PCR products. In thisexperiment, the DNA samples were run through the PCR protocol and thenelectrophoresed in on a 2% SeaKem GTG agarose (FMC BioProducts). In mostexperiments, a λ-HindIII digest (Sigma) was used as a marker (usually inlane 1). In other experiments, the "Biomarker--low" obtained fromBioVentures was used as a marker lane. The gel was then stained withethidium bromide (0.5 μg/l) and DNA bands were observed using a model6-63 transilluminator (UVP).

For Salmonella, an internal probe, "TS21" with the sequence5'-TACATCGTAAAGCACCATCGCAAAT-3' (SEQ ID NO:5) chosen from among a largepreviously published sequence, was then used to confirm the identity ofthe PCR products. For Shigella, two internal probes, with the sequences5'-AGCAGTCTTTCGCTGTT-3 (SEQ ID NO:8) and 5'-AAACGCATTTCCTTCAC-3' (SEQ IDNO:9) were used interchangeably as probes. These Shigella sequences wereused both as probes and as primers in a nested primer PCR protocol todetect Shigella. These sequences were chosen from among a largepreviously published sequence (A. B. Hartman et al., "Sequence andmolecular characterization of a multicopy invasion plasmid antigen gene,ipaH, of Shigella flexneri, J. Bacteriol., 172:1905-1915 [1990]).

The probe oligonucleotides were 3' labeled with digoxigenin-11-dUTP,using a Genius 5 nonradioactive DNA labeling kit (Boehringer Mannheim)per the manufacturer's directions. PCR products were transferred ontoHybond-N+ positively charged nylon membranes (Amersham) using a PosiBlotpressure blotter (Stratagene) for Southern analysis (E. M. Southern,"Detection of specific sequences among DNA fragments separated by gelelectrophoresis," J. Mol. Biol., 98:503-517 [1975]). DNA was fixed tothe nylon membranes by UV irradiation at 254 nm for 2 min using aCL-1000 ultraviolet crosslinker (UVP).

Hybridization was performed at 55° C. in presence of a labeledoligonucleotide internal probe (2 pmol/ml). The hybridized filters werewashed twice with a high-salt solution (2×SCC in 0.1% SDS) at 50° C. AGenius 3 nucleic acid detection kit (Boehringer Mannheim) was used toprepare the hybridized filters for chemiluminescent detection. Thehybridization signals were visualized on X-OMAT (Eastman Kodak) usingautoradiography as described by Sambrook et al., Molecular Cloning: ALaboratory Manual, 2d ed., Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y. (1989).

EXAMPLE 5 OPTIMIZATION OF PCR CONDITIONS

A series of experiments were conducted in which various parameters weretested in the PCR protocol to fine-me the procedure to amplifySalmonella, but not E. coli DNA.

The template DNA was obtained from Salmonella or E. coli as described inthe previous examples. In these experiments, a λ-HindIII digest (Sigma)was included as a marker. All samples were processed in a single (i.e.,not nested) primer PCR for thirty cycles, although at varyingtemperatures as described below:

Experiment 1

In this experiment, two S. typhimurium isolates (lanes 2 and 3), S.heidelberg (lane 4), S. bredeney (lane 5), E. coli 890 (lane 7), E. coli894 (lane 8), E. coli 889 (lane 9), and E. coli S1-3 (lane 10) weretested. Lane 1 contained the λ-HindIII ("HindIII") digest as marker. Thetwo primers, BR-SALb and BR-SALa were used in each of these experiments.

First, low annealing temperatures were tested. Following the initial 94°C. 5 minute exposure, the samples were cycled at 94° C. for 2 min., 53.5for 1 min., and 72.0° C. for 1 min. At these cycling conditions, all ofthe samples were amplified. These results are shown in FIG. 3, with thearrow showing the band of interest. As shown in this figure, there aremultiple bands present in the HindIII lane, (lane 1) as well as multiplebands in the Salmonella lanes (lanes 2-9), and PCR product present inthe E. coli lanes (lanes 8-10; very faint bands appear to be barelyvisible in lanes 9-10).

Experiment 2

In the next run, the cycling parameters were adjusted to 94° C. for 2min., 55.5° C. for 1 min., and 72.0° C. for 1 min. The same laneconfiguration was used as described above for Experiment 1. As shown inFIG. 4, these conditions resulted in the production of multiple bands inthe HindIII lane, as well as multiple bands in the Salmonella lanes, andvery faint PCR products present in the E. coli lanes as well.

Experiment 3

Because these low annealing temperatures produced unsatisfactoryresults, higher temperatures were then tested. The same laneconfiguration was again used, with the exception that lanes 9-10 wereempty. In this run, the mixtures were exposed to thirty cycles of 94° C.for 2 min., 56.5° C. for 1 min., and 72.0° C. for 1 minute. The PCRproducts were then run on gels and visualized. FIG. 5 shows the resultsfor this experiment. As shown in this Figure, although there was goodamplification of Salmonella there also was detectable amplification ofsome E. coli strains, again indicating that the reaction conditions werenot sufficiently stringent to produce specific results.

Experiment 4

Higher temperatures were then used in the PCR, in an attempt to increasethe specificity of the reaction. In this run, the samples were exposedto 30 cycles of 94° C. for 2 min., 58.5° C. for 1 min., and 72.0° C. for1 min. As described above, the reaction products were run on gels andvisualized, with the same lane configurations as described in Experiment3. As shown in FIG. 6, there was no amplification of Salmonella nor E.coli, indicating that the reaction conditions were too stringent toproduce results. The only band observed was present in the controlHindIII lane.

Experiment 5

Next, the cycling temperatures were again changed used in the PCR, in anattempt to provide optimal sensitivity and specificity in the reaction.In this run, the samples were run in 30 cycles of 94° C. for 2 min.,57.0° C. for 30 sec., and 72.0° C. for 1 min. Again, as described above,the reaction products were run on gels and visualized, with the samelane configurations as described in Experiment 3. As shown in FIG. 7,there was good amplification of Salmonella, but no amplification of E.coli. However, the bands produced by the Salmonella PCR products werenot clear and sharply distinct.

Experiment 6

In a final run, the annealing temperature was again changed, in anattempt to provide optimal sensitivity and specificity in the reaction.In this run, the samples were run for 30 cycles of 94° C. for 2 min.,57.5° C. for 30 sec., and 72.0° C. for 1 min. As described above, thereaction products were mn on gels and visualized with the same laneconfigurations as described in Experiment 3. As shown in FIG. 8, therewas good amplification of Salmonella, with sharp and distinct bands, butwith no amplification of E. coli. Thus, these conditions appear optimalfor amplification of Salmonella DNA, without amplifying E. coli DNA aswell.

EXAMPLE 6 SEEDING OF SEWAGE AND SLUDGE WITH SALMONELLA

Various isolates were used in seeding experiments, in whichenvironmental waters such as sewage and sludge were spiked with knownquantities of organisms. For these seeding experiments, mid-log-phaseSalmonella typhimurium (ATCC 14028) were collected, washed twice withphosphate-buffered saline, and serially diluted (10⁻² to 10⁻⁹). Platecounts (see e.g., Standard Methods, pp. 886-889) were performed on theseserial dilutions spread on Hektoen enteric agar (Difco), to determinethe number of viable Salmonella in the sample.

The serial dilutions were then seeded into samples of primary treatedsewage. Nine ml of sewage were placed in each of six tubes. To these sixtubes, 1 ml of diluted organism suspension was added. The samples weremixed well and filtered through a Swinnex filter fitted with an FHLPfilter (Millipore). The filtrate was then placed in a microfuge tube, towhich 500 μl of sterile water was added. The samples were placed at 85°C. for 5 min., vortexed, and then freeze-thawed. In the freeze-thawsequence, samples were first frozen in dry ice for five minutes, thenthawed at 65° C. for five minutes. This cycle was repeated six times.Samples were then placed in a Sephadex G-200 column and spun at 2000 xgfor 5 minutes. Portions of the eluent were then used in PCR as describedin Example 5, Experiment 6, run in gel electrophoresis and the bandsvisualized as described above (using the primers BR-SALa and BR-SALb).

FIG. 9 shows the results obtained for these dilutions. Lane 1 containedmarker DNA (BioVentures) (with bands corresponding to 1114, 900, 692,501, 489, 404, 320, 240, 190, 147 and 124). Lane 2 was a control whichcontained sewage only, with no added organisms (i.e., an unseededcontrol). Lanes 3 through 9 contained dilutions 10⁻², 10⁻³, 10⁻⁴, 10⁻⁵,10⁻⁶, 10⁻⁷, 10⁻⁸ and 10⁻⁹ respectively. Lane 10 was a control containing100 CFU, and no sewage. As shown in the figure, amplifiable DNA waspresent in the lower dilutions (lanes 3, 4 and 5). Southern blots wereperformed using standard methods (E. M. Southern, "Detection of specificsequences among DNA fragments separated by gel electrophoresis," J. Mol.Biol., 98:503-517 (1975) and confirmed these results.

Primary treated sewage obtained from the Sand Island facility was alsoused in experiments designed to demonstrate direct detection ofSalmonella in sewage.

EXAMPLE 7 PCR OF ENVIRONMENTAL SAMPLES

Samples obtained from environmental and sewage effluents processed asdescribed above were tested using the optimal PCR conditions describedin the above example and run on a gel. FIG. 10 shows the resultsobtained. Lane 1 contains marker DNA (BioVentures); Lane 2 contains isfrom Manoa Stream, Lanes 3 and 4 contain sewage effluent, Lane 5, 6 and7 contain ocean water, Lane 8 is a negative control, and lane 9 is apositive control. As shown, the only positive band was observed with thepositive control, indicating that no Salmonella was present in any ofthese environmental samples.

EXAMPLE 8 NESTED PRIMER PCR PROTOCOL FOR SALMONELLA

In addition to the PCR protocol using a single primer pair, a nested PCRprotocol was developed. In this nested primer protocol, the first PCRstep subjected to samples to 35 cycles, each consisting of 120 sec. at94° C., 20 sec. at 57.5° C., and 60 sec. at 72° C., followed by 7 min.at 72° C. to complete synthesis. In the first PCR step primers BR-SALand BR-SALa and BR-SALb were used. The second nested PCR step (usingprimers SAL-Ia & SAL-Ib) was carried out in a total volume of 100 μl,with a 2 μl sample of the first round of PCR used as the template. Thecycle profile consisted of denaturation at 95° C. for 5 min followed bytwenty cycles, each consisting of 30 sec. at 94° C., 60 sec. at 54° C.,and 60 sec. at 72° C., followed by completion of synthesis at 72° C. for10 min. The PCR products were analyzed by gel electrophoresis asdescribed above. These results are shown in FIG. 11. In this Figure,Lane 1 contains marker; Lane 2 contains 200 pg; Lane 3 contains 20 pg;Lane 4 contains 2 pg; Lane 5 contains 200 fg; Lane 6 contains 20 fg;Lane 7 contains 2 fg; Lane 8 contains 200 ag; Lane 9 contains 20 ag; andLane 10 contains control.

EXAMPLE 9 PCR OF SHIGELLA

Following the fine-tuning of the Salmonella PCR assay, a parallel assaywas developed for rapid detection of Shigella species.

Multiple primers were developed, including primer ShigA (SEQ ID NO:6)and ShigB (SEQ ID NO:7), which were prepared from a previously publishedShigella restriction fragment sequence (Hartman et al., "Sequence andmolecular characterization of a multicopy invasion plasmid antigen gene,ipaH, of Shigella flexneri, J. Bacteriol., 172:1905-1915 [1990]). Theseprimers are shown in Table 6. The primers were produced with anautomatic DNA synthesizer (ABI 381A; Applied Biosystems).

                  TABLE 6                                                         ______________________________________                                        Primers Used for Detection of Shigella                                        Primer   Sequence          SEQ ID NO:                                         ______________________________________                                        ShigA    5'-TTG ACC GCC TTT                                                                              SEQ ID NO: 6                                                CCG ATA C-3'                                                         ShigB    5'-ACT CCC GAC ACG                                                                              SEQ ID NO: 7                                                CCA TAG A-3'                                                         ______________________________________                                    

These primers were designed such that they are compatible for use incombination with the Salmonella detection assays, while retaining thesensitivity and specificity of both assay systems.

As a control to determine whether these primers would producecross-reactivity, all of the Shigella and non-enteric strains listed inTable 3 were tested using these primers and PCR testing protocols. TheDNA extracts were prepared as described above in Example 2 forSalmonella, and the PCR cycle conditions were the same as those used inExperiment 6 of Example 3. The sequence shown in SEQ ID NO:8 was used asa probe to detect the presence of Shigella bands in gel electrophoresis.The same gel electrophoresis procedures as were used above forSalmonella were also used in this Example, with the exception that theShigella probe was used. These data are not shown, but indicated thatthere was specific and sensitive amplification of Shigella DNA, withoutany problems associated with cross-reactivity.

FIG. 12, shows the results obtained for various dilutions S. dysenteriaeDNA. In this figure, lane 1 was a molecular weight marker(BioVentures)(1000 bp, 700 bp, 525 bp, 500 bp, 400 bp, 300 bp, 200 bp,100 bp, and 50 bp). Lane 2 contained 100 ng of S. dysenteriae genomicDNA; lane 3 contained 10 rig; lane 4 contained 1 ng; lane 5 contained0.1 ng; lane 6 contained 100 pg; lane 7 contained 10 pg; lane 8contained 1 pg; lane 9 contained 0.1 pg; lane 10 contained 100 fg; andlane 11 contained 10 fg.

As shown in this figure, good amplification was obtained with as littleas 100 pg of S. dysenteriae DNA, and a detectable band was observed at10 pg DNA.

EXAMPLE 10 NESTED PRIMER PCR PROTOCOL FOR SHIGELLA

In addition to the PCR protocol using a single primer pair, a nested PCRprotocol was developed. In this nested primer protocol, the first PCRstep subjected to samples to 35 cycles, each consisting of 120 sec. at94° C., 20 sec. at 57.5° C., and 60 sec. at 72° C., followed by 7 min.at 72° C. to complete synthesis. In the first PCR step, primers ShigAand ShigB were used. The second nested PCR step (using two additionalprimers, "Shig-Ia" and "Shig-Ib," as shown in the following Table), wascarried out in a total volume of 100 μl, with a 2 μl sample of the firstround of PCR used as the template. The cycle profile consisted ofdenaturation at 95° C. for 5 min followed by twenty cycles, eachconsisting of 30 sec. at 94° C., 60 sec. at 54° C., and 60 sec. at 72°C., followed by completion of synthesis at 72° C. for 10 min. The PCRproducts were analyzed by gel electrophoresis as described above(results not shown).

                  TABLE 7                                                         ______________________________________                                        Nested Primers Used for Detection of Shigella                                 Primer   Sequence          SEQ ID NO:                                         ______________________________________                                        Shig-Ia  5'-ACG AGT CTT    SEQ ID NO: 8                                                TCG CTG TT-3'                                                        Shig-Ib  5'-AAA CGC ATT    SEQ ID NO: 9                                                TCC TTC AC-3'                                                        ______________________________________                                    

EXAMPLE 11 MULTIPLEX PCR AMPLIFICATION FOR SIMULTANEOUS AND SPECIFICDETECTION OF SALMONELLA AND SHIGELLA

In this experiment, specific DNA fragments from genomic DNAs of S.dysenteriae (ATCC #11456b) and S. typhimurium (ATCC #14028) weredetected in a multiplex PCR system. The primers used in this experimentwere the two Shigella primers (ShigA and ShigB) and the Salmonellaprimers designated BR-SALa and BR-SALb. The PCR reaction mix contained100 ng of DNA from both organisms and 0.3 μM of each primer. The sampleswere run for 30 cycles of 94° C. for 2 min., 57.5° C. for 20 sec., and72.0° C. for 1 min. As shown in FIG. 13, varying MgCl₂ concentrationswere tested in this experiment. The reaction products were run on gelsand visualized as described in the previous examples. In FIG. 13, Lane 1contains molecular weight markers sized at 1000, 700, 525, 500, 400,300, 200, 100, and 50 base pairs. Lane 2 shows simultaneousamplification of both Salmonella and Shigella with 40 mM MgCl₂. Lane 3shows simultaneous amplification of both Salmonella and Shigella with 35mM MgCl₂. Lane 4 shows simultaneous amplification of both Salmonella andShigella with 30 mM MgCl₂. Lane 5 shows simultaneous amplification ofboth Salmonella and Shigella with 25 mM MgCl₂. Lane 6 shows simultaneousamplification of both Salmonella and Shigella with 20 mM MgCl₂. Lane 7shows simultaneous amplification of both Salmonella and Shigella with 15mM MgCl₂. Control lane 8 shows the 408 bp specific S. dysenteriae PCRproduct, and lane 9 shows the 525 bp specific S. typhimurium PCRproduct. The reaction mixture in these lanes was the usual composition,as described above.

Based on this experiment, it appears that good, specific amplificationof S. dysenteriae and S. typhimurium were achieved at MgCl₂concentrations ranging from 40-30 mM, with acceptable results observedwith concentrations ranging from 25-20 mM. At 15 mM, the S. typhimuriumfragment was virtually undetectable, although the S. dysenteriaefragment was readily apparent. Thus, conditions including 40 mM MgCl₂appear optimal for the simultaneous amplification of S. typhimurium andS. dysenteriae DNA.

From the above, it is clear that the present invention provides themethods and compositions for the rapid, yet specific detection andidentification of enteric organisms from various types of samples,including environmental and sewage samples. In addition, the presentinvention also provides the compositions and methods needed for therapid definitive diagnosis of these pathogens. Indeed, the presentinvention clearly provides the compositions and methods needed for therapid definitive diagnosis/detection/identification of two of the mostimportant waterborne pathogens. Of particular importance is the factthat the present invention avoids problems associated with interferingsubstances often present in environmental samples, which may affect theamplification and/or detection of organisms.

It is not intended that the present invention be limited to a particularsample type. For example, it is contemplated that the present inventionwill be useful in various settings, including clinical, public health,and veterinary laboratories, as well as water quality, food and dairylaboratories.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 9                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       ACGGTTGTTTAGCCTGATAC20                                                        (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       CTGGATGAGATGGAAGAATG20                                                        (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       GTTCGGCATTGTTATTTCT19                                                         (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       CTCAGGGTCATCGTTATTC19                                                         (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       TACATCGTAAAGCACCATCGCAAAT25                                                   (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       TTGACCGCCTTTCCGATAC19                                                         (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       ACTCCCGACACGCCATAGA19                                                         (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       AGCAGTCTTTCGCTGTT17                                                           (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       AAACGCATTTCCTTCAC17                                                           __________________________________________________________________________

What is claimed is:
 1. A composition comprising one or more isolated andpurified nucleic acid sequences selected from the group consisting ofSEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ IDNO:6, SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9.
 2. The composition ofclaim 1, wherein the isolated and purified nucleic acid selected fromsaid group hybridizes to Salmonella nucleic acid to form an amplifiablereaction mixture.
 3. The composition of claim 1, wherein the isolatedand purified nucleic acid selected from said group hybridizes toShigella nucleic acid to form an amplifiable reaction mixture.